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AMER. ZOOL., 26:769-780 (1986)
The Structure and Organization of Genetic Material
1
MONROE W. STRICKBERGER Department of Biology, University of Missouri—St. Louis, St. Louis, Missouri 63121
SYNOPSIS. Some of the major features of nucleic acid structure and replication are reviewed in respect to their constancy and variability. These two qualities of conservation and change are broadly discussed in terms of the roles that genetic informational molecules play in biological evolution.
INTRODUCTION There are perhaps two approaches in teaching the structure of genetic material as a process of discovery; that is, in explor- ing this topic in the context of our sym- posium, "Science as a Way of Knowing." One approach, that of describing why and how genetic material was historically dis- covered by biologists, has already been par- tially provided for you by Dr. Moore in the essay that accompanies this symposium, and is further discussed in various genetics textbooks, and in the more thorough accounts of Judson (1978), Olby (1974), Portugal and Cohen (1978), Watson (1968), and many others. The second approach, to try to understand and explore the role that genetic material occupies in biology because of evolutionary changes in its structure and organization, can be as inter- esting for some students as the more his- torical approach, and is one to which this paper is mostly devoted. The cornerstone of this discussion is that genetic material provides to all of biology two basic qualities that enable evolutionary changes to occur: constancy and variability. Constancy provides the basis for our fun- damental genetic observation that "like produces like," and variability provides the basis for change in the sense that "like also produces unlike." If we define evolution as changes in biological information over time, it is clear that evolution must entail changes (variability) in the duplication (constancy) of biological information. Since
(^1) From the Symposium on Science as a Way of Know- ing — Genetics presented at the Annual Meeting of the American Society of Zoologists, 27—30 December 1985, at Baltimore, Maryland.
both of these qualities derive from the pro- cesses by which genetic material is copied and transmitted, we can begin with briefly examining the molecular structure of this material and its mode of replication. The genetic material of all terrestrial organisms dating back to the early history of life, four billion or so years ago, has probably been primarily, if not entirely, in the form of nucleic acids (Fig. 1). These are long-chained molecules composed of nucleotide subunits, each containing a pen- tose (5-carbon) sugar, a monophosphate group, and a nitrogenous base. The two kinds of sugar used in nucleic acids, ribose (hydroxylated at the 2' carbon position) and deoxyribose (lacks 2' hydroxyl) provide the names for the two kinds of nucleic acids, ribonucleic acid (RNA) and deoxyribonu- cleic acid (DNA). The phosphate groups occupy the same position in both nucleic acids, serving to tie the 3' carbon of one sugar to the 5' carbon of its neighbor via a phosphodiester bond. Connected to the 1' carbon of each sugar is one of four kinds of nitrogenous heterocyclic bases, two of which are purines (adenine [A] and gua- nine [G] in both DNA and RNA) and two are pyrimidines (cytosine [C] and thymine [T] in DNA, and cytosine [C] and uracil [U] in RNA). It would seem that the restriction of nucleic acid composition to only four dif- ferent kinds of bases would limit the mes- sage-bearing capacity of these molecules to only four kinds of messages; but this is, of course, not so. The fact that nucleic acid molecules may be many thousands or mil- lions of nucleotides long, and that each message can be encoded by a unique linear sequence of nucleotides, endows these molecules with the capacity to bear an
769
770 MONROE W. STRICKBERGER
(a) polynucleotide chain structure (DNA and RNA) 5'end
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_ Base
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CH, (^) HO I © ©^13 C
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pyrimidines, one-ring bases:
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purines, Iwo-ring bases (DNA a n d RNA):
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H guanine ( 0 )
FIG. 1. Structural formulae for nucleic acids and their components, (a) Structure of a tetranucleotide sequence showing the general positioning within a nucleotide of the phosphate group, the sugar moiety, and the nitrogenous base, as well as the phosphodiester bonds that connect nucleotides together, (b) The two kinds of sugars found in cellular nucleic acids, (c) The common bases and their designations.
immense variety of immensely complex messages. For any one nucleotide position, 4 different messages are possible (A, G, C, or T); for two nucleotides in tandem, 16 different messages are possible (AA, AG, AC, AT, GG, GC,.. .); and so on: the rule being simply that for a linear sequence of
n nucleotides, 4" different possible mes- sages can be encoded. Thus a linear sequence of only 10 nucleotides can be used to discriminate between more than one million potentially different messages. In reality the biological messages that nucleic acids carry are mostly translated
772 MONROE W. STRICKBERGER
replicating I DNA:
protein:
FIG. 3. Diagram of how sequential information is transferred between the three major biomolecular polymers, DNA, RNA, and protein. Among the cel- lular choices are to replicate a DNA molecule by means of DNA polymerase enzymes to produce two identical double strands, or to transcribe one strand of a DNA molecule into RNA by means of RNA polymerase enzymes. Transcribed RNA can then act as a "mes- senger" which is translated by ribosomal machinery into a polypeptide chain that then folds to form a protein or protein component. The nucleotide sequence on messenger RNA is exactly complemen- tary to its DNA template, and a triplet of three nucleo- tides (triplet codon) on the messenger specifies one amino acid.
in the DNA backbone, and changes in dis- tance between adjacent phosphate groups, which then, in turn, modify the nucleic acid helical conformation (see Saenger, 1984). The replicatory power of the DNA dou- ble helix obviously derives from the ability of each of the two strands to serve as a template for a newly complementary strand, so that two new double helices can
be formed bearing nucleotide sequences that are identical to each other and to that of the parental molecule (Fig. 3). For myself and many others, it was Meselson and Stahl's (1958) experimental support for such "semiconservative" replication that brought the Watson-Crick DNA model past the stage of being merely a very attractive hypothesis. To briefly summarize so far, the struc- ture of nucleic acids encodes information by means of the linear placement of four different kinds of nucleotides. This infor- mation can be replicated via polymerase enzymes; and can be used biologically, mostly by translation into polypeptides. In cells, sequential information in DNA is transmitted to other cells by replication, thus providing the "genotype." The expression of this information, the "phe- notype," begins primarily with the tran- scription of this sequential information into the various kinds of RNA (messenger, ribo- somal, transfer), followed by the subse- quent translation of messenger RNA into amino acid sequences, and then by all of the various interactions that characterize the organism.
THE PERENNIAL "GOLDEN AGE" This one-way direction for transfer of molecular sequential information, nucleic acid to protein, prompted Crick (1958) to depict this as the "Central Dogma," and it seemed as though an understanding of all basic genetic molecular features would soon be at hand. In fact, within a decade, by the late 1960s, some molecular biologists sug- gested that the "Golden Age" of progress in biology was coming to an end, and there were probably very few real surprises in store (Stent, 1969). Nevertheless, depend- ing on how easily one is surprised, inves- tigations into the structure of genetic material continue to offer unforeseen nov- elties which help to provide an apprecia- tion of how extensive are both the vari- ability and the change experienced by these basic molecules. Among such "surprises," have been the following:
- The discovery that the transmission of sequential information occurs not only in the direction of DNA -> RNA, but also
STRUCTURE OF GENETIC MATERIAL 773
TGTGTG G ACACAC C
AATTGT TTAACA|C|T1C|G|C|C|T|A|T|TGTTAA|A|GTGTGT
ACAATT T
FIG. 4. Nucleotide sequence of the E coll operator region at which the lac repressor attaches that con- trols the synthesis of enzymes involved in lactose metabolism. The symmetry of this region is "palin- dromic" because the sequences in the boxes to the left of the axis of symmetry are inverted repeats of the boxes to the right of this axis. (After Strickberger, 1985.)
in the direction RNA — DNA. Such "reverse transcription," catalyzed by the enzyme reverse transcriptase, is the mode by which RNA retroviruses incorporate their genetic material into the DNA of host chromosomes (see, for example, Weiss et al, 1982).
- The discovery that DNA sequences carry not only information expressed through transcription and translation, but also information that determines the con- formation of the molecule itself. For exam- ple, "palindromic" sequences which pre- sent the same nucleotides (but with inverted order) on either side of a central axis are used in some genes as part of the recog- nition sites ("promoters") for enzyme attachments that help initiate transcrip- tion, or used as sites for the attachment of repressors that prevent transcription (Fig. 4). These and other special DNA sequences such as "inverted repeats" can produce loops, twists, and kinks that allow inter- action between DNA and various enzymes, viruses, and plasmids, and also provide loci at which mutation rates are preferentially increased or decreased (see, for example, Miller, 1983). In eukaryotes, DNA is orga- nized into nucleosomal structures using histone proteins, and specific nucleotide sequences undoubtedly also play a role as recognition sites that help activate nucleo- some placements or displacements (see Eis- senberg et al., 1985).
- The discovery that DNA may be coiled in a left-handed rather than right- handed direction (Fig. 5). Such left-handed double helical sections, called "Z-DNA"
FIG. 5. Space-filling models of B-DNA and Z-DNA double helical molecules. The heavy lines used to con- nect the phosphate groups in each chain show the zig- zag placement of phosphates in Z-DNA in contrast to the smoother curve of their relationship in B-DNA. The "major" and "minor" grooves in B-DNA differ in depth but do not extend to the central axis of the molecule, whereas the indicated Z-DNA groove pen- etrates the axis of the double helix. (After Rich et al, 1984.)
because of their zig-zag placement of phos- phate groups, were first associated with alternating CG sequences but are now also observed in other sequences as well. Like some other distortions of the DNA mole- cule, Z-DNA is believed to occur within particular chromosomal regions at which regulatory activity takes place (Rich et al., 1984).
- The discovery of DNA sequences in eukaryotes that are repeated many times over (Table 1). Some of these repeated sequences are very short, and are localized to specific chromosomal regions such as centromeres, whereas others of somewhat longer lengths are dispersed throughout the genome. In mammals, among several families of dispersed sequences that have been identified, is the Alu family found in primates, represented by perhaps more
STRUCTURE OF GENETIC MATERIAL (^775)
- The discovery of "split" eukaryotic genes in which amino acid-coding nucleo- tide sequences called "exons" are inter- rupted by noncoding sequences called "introns" (Fig. 6). The sequences in these split genes are transcribed from DNA to RNA, and the RNA is then "processed" so that the introns are removed and the exons spliced together. Such processed mature messenger RNA molecules are then transported across the nuclear membrane to the cytoplasm to be translated into poly- peptides. Whatever the advantages of intron-exon structures, these seem suffi- ciently great to account for the fact that gene splitting occurs in almost all verte- brate protein-coding genes so far exam- ined, as well as in many similar genes in other eukaryotes. Gilbert (1979) suggested that each exon may have originally coded for a single polypeptide "domain" that could be used for a specific function, and others such as Blake (1985) suggested a relationship between the average size of an exon (coding for about 20 to 80 amino acids) and the size of the smallest poly- peptide sequence that could be folded into a stable structure (about 20 to 40 amino acids). Because of the flexibility of exon arrangement and intron removal, the exons coding for these polypeptide subunits could then be combined in various ways to form genes that produced optimal polypeptides. According to Doolittle (1978) and others, cellular organisms ancestral to both pro- karyotes and eukaryotes possessed such intron-exon structures, but these were abandoned in prokaryotic lines because of an increased intensity of selection for "streamlining" DNA replication and tran- scription efficiency.^3
- The discovery of "pseudogenes," defined as nucleotide sequences that are inexact nonfunctional copies of normally active genes (Li, 1983). Pseudogene non- functional status may be caused by one or more mutations that affect transcription (mutated promoters), RNA processing (mutated introns), or translation (nonsense mutations). It was also surprising to find that all introns had been removed from some pseudogenes ("processed genes"), indicating that they had been incorporated into DNA by the reverse transcription of previously processed messenger RNA mol- ecules. In many instances, pseudogenes are found closely associated with their active gene counterparts in "gene clusters," thus helping to provide evidence for the prev- alence of mechanisms such as unequal recombination which is believed to have produced many genes and gene families by duplication.
- The discovery that a single DNA sequence could be used to code for more than one kind of protein; that is, the exis- tence of "overlapping genes." Overlaps of this kind may arise from the use of differ- ent translation initiation sites located along the same set of codons ("same reading frame"), or by using different sets of codons for the same nucleotide sequence ("differ- ent reading frames"). In many small genomes such as bacteriophage <£X (Godson et al., 1978), such overlapping genes seem to serve the purpose of increas- ing the number of different kinds of poly- peptide products from an evolutionarily restricted length of DNA.'
- The discovery that new combinations can be formed between fairly widely dis- persed DNA sequences within vertebrate
(^3) It is not clear why selection for "streamlining" should affect prokaryotes more than eukaryotes, espe- cially since there are (and were) many single-celled eukaryotes (and even multicellular organisms) in which "streamlined" metabolic and reproductive mecha- nisms would be as adaptive as in single-celled prokary- otes. An alternative explanation for eukaryotic introns may lie in the necessity for messenger RNA transport across the nuclear membrane, and the role that some of the small nuclear ribonucleoprotein particles (snRNP) play in both intron excision and messenger RNA transport. Perhaps eukaryotic cells became reliant on both these functions of snRNPs once nuclear
membranes had evolved and messenger RNA mole- cules had to be carried into the cytoplasm. Certainly, the ubiquitousness of introns in eukaryotic genes, the considerable expense in maintaining these split arrangements, and their absence in nontranslated "processed pseudogenes" (see below), indicates a function for introns beyond that of merely separating polypeptide domains that no longer need to be sep- arated. A different explanation offered by Cech (1985) is that introns may persist because their excision from DNA molecules is rarely, if ever, exact, and they must therefore be kept intact to prevent the mis-splicing of exons. Thus intron DNA is primarily preserved because it must be transcribed into "selfish RNA"!
(^776) MONROE W. STRICKBERGER
lit!? P 0 2 5 0 5 0 0 7 5 0 1000 1250 1500 scale I^ I^ I^ I^ I^1 I inl in
ex? | transcription I
ox
RNA" J A A A A A A A A A A A A A A A A / '
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0 globin amino acids
FIG. 6. The intron-exon structure for nucleic acid sequences involved in the production of the human /3-globin polypeptide chain, 146 amino acids long, one of the components of normal adult hemoglobin. The top portion of the diagram indicates the approximate 1,500 nucleotide base-pair length in the /3-globin DNA sequence, and the structure of this sequence in terms of two introns (inl, in2) and three exons (exl, ex2, ex3). Intron 1 is 130 nucleotides long, and separates codons that will later be translated into amino acids numbered 30 and 31 on the j3-globin chain. Intron 2, 850 nucleotides long, separates codons for/3-globin amino acids numbered 104 and 105. Although the entire DNA sequence is transcribed into messenger RNA, the introns are precisely removed by special RNA processing in the nucleus, and the exon sequences are spliced together and then translated
somatic cells to form unique antibody-pro- ducing genes. This mechanism is a some- what different evolutionary answer to the problem described above for increasing the number of proteins in smaller genomes. In this case, selection from an array of many millions of different possible kinds of anti- body proteins can occur on demand pro- duced by only a limited number of DNA sequences (Honjo and Habu, 1985).
- The discovery that genes can directly modify the DNA sequences of other genes within the same genome. Whereas normal recombinational mechanisms that account for the exchange of sequences between homologous chromosomes are usually per- formed reciprocally (e.g., ABC x abc -* ABc + abC), a significant number of events have been identified that can only be explained as the result of nonreciprocal exchange (e.g., ABC x abc - ABc + aBC). That is, a DNA sequence on a donor gene is directly adopted by a recipient gene with- out any modification of the donor. Such instances have been subsumed under the titles "gene conversion" (Nagylaki and Petes, 1982), "concerted evolution" (Arnheim, 1983), and "molecular drive" (Dover, 1982), and seem to result from variations in replication and recombina- tion mechanisms. They help to explain the fact that certain repetitive DNA sequences are remarkably alike within a species, but can show significant differences between species. Apparently, these devices can "homogenize" some repetitive sequences within species so they resemble only one such sequence.
NUCLEIC ACID EVOLUTION In addition to the more classical mech- anisms of genetic change (e.g., base substi- tutions, deletions, duplications, inversions, insertions, etc.), the various further com-
into a continuous /3-globin amino acid sequence. At the bottom of this figure, a single /3-globin polypeptide chain has been diagrammatically split to indicate the three subcomponents ("domains") respectively pro- duced by the three exon nucleotide sequences. (From Strickberger, 1985, with additions.)
778 MONROE W. STRICKBERGER
50 A
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c-u FIG. 7. Nucleotide sequence and secondary structure of the "midivariant-1" RNA molecule found after selection for rapid replication among Q/3 genomes in mixtures in which RNA replicase enzymes are provided. About 95 percent of the Q0 genome has been eliminated because of this selection, and the remaining 221 nucleotides of midivariant-1 are apparently only those necessary to enable the replicase enzyme to bind to the molecule so that replication can occur. (After Miele et at, 1983.)
complementary "messenger" strands for this purpose. Three separate functional classes of nucleotide sequences thus even- tually arose, storage, messenger, and trans- lational (ribosomal and transfer). These were all probably RNA, since this nucleic acid is still used for two of these purposes in all organisms (messenger and transla- tional), and is also used for genetic storage purposes in some viruses that replicate their genetic material directly by means of RNA replicases. In addition, some RNA mole- cules such as transfer RNA and RNAs used in RNA processing still preserve catalytic properties, indicating that this nucleic acid may well have provided both information and some enzyme-like activity. Cellular difficulties caused by attempting
to maintain RNA molecules with two prim- itive but quite different functions, infor- mation storage and protein translation, would have offered advantages to organ- isms that could utilize a different nucleic acid for storage purposes, DNA. The enzymes that translate RNA into protein do not function with DNA, thus permitting the more uniformly structured double- helical DNA to be restricted exclusively to the storage of information and to the tran- scription of one of its strands to form mes- senger RNA. It has also been proposed that DNA genetic material would offer a more easily protected molecule than RNA because the 2' hydroxyl group in the ribose sugar of RNA causes it to undergo more rapid hydrolytic cleavage than the 2' de-
STRUCTURE OF GENETIC MATERIAL 779
oxyribose of DNA. A third suggested rea- son for a change in genetic mterial is that RNA replication is more error-prone than DNA replication because RNA polymer- ases do not possess the exonuclease editing functions of DNA polymerase. Further- more, the transition from RNA to DNA as the genetic material may have simply entailed the presence of reverse transcrip- tase enzymes. These enzymes, perhaps originally involved only in RNA replica- tion, could later have been used to transfer genetic information from RNA to DNA.
CONCLUDING REMARKS From everything discussed so far, I believe it is clear that the basic biological and evolutionary qualities of constancy and variability derive ultimately from the rep- lication process utilized by nucleic acids. Faithful replication of nucleotide sequences has provided constancy, and errors and changes in nucleic acid replication have provided variability. However, although nucleic acids are essential to biological pro- cesses, it is also important to recognize that replication includes further higher orders of biological organization, in the sense that organisms, populations, and communities possess various nonmolecular qualities that are also replicated, such as sexual, social, and cultural behaviors, geographical and ecological distributions, populational and species interactions, etc. In the selection of a balance between constancy and variability at all levels, we can see that living processes deal with the present and the future by making use of a rather remarkable hindsight. That is, although organisms have no way of pre- dicting the exact future, they have, through their evolutionary experience, visualized the past. To the extent that the past pro- vides a reasonable picture of the future (even past uncertainties become a picture of future uncertainties), evolution can be said to have provided informational vision that can often dimly see the future. The fact that biological processes have survived for so long is certainly testimony to the foresight provided by historical genetic information. On the whole, it seems both valid and
valuable to look at life as a series of pro- cesses rather than as one or more material entities. Certainly the preservation of life is not through the immortality of its com- ponents, since all living organisms and their organic components eventually "die." Rather, life is preserved in the sense that the information that allows living processes to persist is continually replaced by repli- cation. It is this information, borne by genetic material and embodied in all of its many interactions, that carries with it the important biological features of constancy and variability.
REFERENCES Arnheim, N. 1983. Concerted evolution of multi- gene families. In M. Nei and R. K. Koehn (eds.), Evolution ofgenes and proteins, pp. 3 8 - 6 1. Sinauer Associates, Sunderland, Massachusetts. Blake, C. C. F. 1985. Exons and the evolution of proteins. Int. Rev. Cytol. 93:149-185. Brown, W. M. 1983. Evolution of animal mitochon- drial DNA. In M. Nei and R. K. Koehn (eds.), Evolution ofgenes and proteins, pp. 6 2 - 8 8. Sinauer Associates, Sunderland, Massachusetts. Cech, T. R. 1985. Self-splicing RNA: Implications for evolution. Int. Rev. Cytol. 93:3-22. Crick, F. H. C. 1958. On protein synthesis. Symp. Soc. Exp. Biol. 12:138-163. Dawkins, R. 1976. Theselfishgene. Oxford Univ. Press, New York. Doolittle, W. F. 1978. Genes-in-pieces, were they ever together? Nature 272:581. Dover, G. 1982. Molecular drive: A cohesive mode of species evolution. Nature 299:111-117. Eigen, M. 1983. Self-replication and molecular evo- lution. In D. S. Bendall (ed.), Evolution from mol- ecules to man, pp. 105-130. Cambridge Univ. Press, Cambridge. Eissenberg.J. C, I. L. Cartwright, G. H. Thomas, and S. C. R. Elgin. 1985. Selected topics in chro- matid structure. Ann. Rev. Genet. 19:485-536. Gilbert, W. 1979. Introns and exons: Playgrounds of evolution. In R. Axel, T. Maniatis, and C. F. Fox (eds.), Eucaryotic gene regulation, p p. 1-12. Academic Press, New York. Godson, G. N., J. C. Fiddes, B. G. Barrell, and F. Sanger. 1978. Comparative DNA sequence anal- ysis of the G4 and 0X174 genomes. In D. T. Denhardt, D. Dressier, and D. S. Ray (eds.), The single-stranded DNA phages, pp. 51-86. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Honjo, T. and S. Habu. 1985. Origin of immune diversity: Genetic variation and selection. Ann. Rev. Biochem. 54:803-830. Jelinek, W. R. and C. W. Schmid. 1982. Repetitive sequences in eukaryotic DNA and their expres- sion. Ann. Rev. Biochem. 51:813-844. Judson, H. F. 1978. The eighth day of creation: Makers
